Abstract

Information on extracellular signals and conditions is often transduced by biological systems using cascades of protein phosphorylation that affect the activity of enzymes, the localization of proteins and gene expression. A model to study signal transduction is the response of the yeast Saccharomyces cerevisiae to osmotic changes as it shares many central themes with information processing modules in higher eukaryotes. Despite considerable progress in our understanding of this pathway, the scale and dynamics of this system have not been addressed systematically yet. Here, we report a comprehensive, quantitative, and time-resolved analysis using high-resolution mass spectrometry of phospho-proteome and proteome changes in response to osmotic stress in yeast. We identified 5534 unique phosphopeptide variants and 3383 yeast proteins. More than 15% of the detected phosphorylation site status changed more than two-fold within 5 minutes of treatment. Many of the corresponding phosphoproteins are involved in the early response to environmental stress. Surprisingly, we find that 158 regulated phosphorylation sites are potential substrates of basophilic kinases as opposed to the classical proline-directed MAP kinase network implicated in stress response mechanisms such as p38 and HOG pathways. Proteome changes reveal an increase in abundance of more than one hundred proteins after 20 min of salt stress. Many of these are involved in the cellular response to increased osmolarity, which include proteins used for glycerol production that is up-regulated to counterbalance the increased osmolarity of the salt containing growth medium. Although the overall relationship between our proteome and published mRNA changes is poor we find an excellent correlation between the subset of osmotic shock up-regulated proteins and their corresponding mRNA changes.

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